1. Field of the Invention
This invention is related to the field of probe-based detection, analysis and quantitation of microorganisms. More specifically, this invention relates to novel PNA probes, probe sets, methods and kits pertaining for the detection of microorganisms. The PNA probes, probe sets, methods and kits of this invention can be used to detect, identify or quantitate one or more organisms in a sample wherein the organisms of interest may include E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas fluorescens or organisms of a bacterial genus including the Salmonella genus, Bacillus genus or Pseudomonas genus.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, probe-based assays have been slow to achieve commercial success. This lack of commercial success is, at least partially, the result of difficulties associated with specificity, sensitivity and reliability.
Hybridization assays hold promise as a means to screen large numbers of samples for conditions of interest. In practice, however, it is often difficult to multiplex a hybridization assay given the requirement that each of the many very different probes in the assay must exhibit a very high degree of specificity for a specific target nucleic acid under the same or similar conditions of stringency. Given the difficulties in specificity, sensitivity and reliability of nucleic acid probes in assays designed to detect a single target nucleic acid, sensitive and reliable methods for the multiplex analysis of samples has been particularly elusive.
Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide which can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. No. 5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). Being a non-naturally occurring molecule, unmodified PNA is not known to be a substrate for the enzymes which are known to degrade peptides or nucleic acids. Therefore, PNA should be stable in biological samples, as well as have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al., J. Am. Chem. Soc. 118:55 44-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, at p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay, appears to be somewhat sequence dependent (Nielsen et al., Anti-Cancer Drug Design 8:53-65, (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)).
Though they hybridize to nucleic acid with sequence specificity (See: Egholm et al., Nature, at p. 567), PNAs have been slow to achieve commercial success at least partially due to cost, sequence specific properties/problems associated with solubility and self-aggregation (See: Bergman, F., Bannwarth, W. and Tam, S., Tett. Lett. 36:6823-6826 (1995), Haaima, G., Lohse, A., Buchardt, O. and Nielsen, P. E., Angew. Chem. Int. Ed. Engl. 35:1939-1942 (1996) and Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and Weiler, K., Nucleosides and Nucleotides 16:1775-1779 (1997) at p 433, col. 1, In. 28 through col. 2, In. 3) as well as the uncertainty pertaining to non-specific interactions which might occur in complex systems such as a cell (See: Good, L. et al., Antisense and Nucleic Acid Drug Development 7:431-437 (1997)). However, problems associated with solubility and self-aggregation have recently been reduced or eliminated (See: Gildea et al., Tett. Lett. 39: 7255-7258 (1998)). Nevertheless, their unique properties clearly demonstrate that PNA is not the equivalent of a nucleic acid in either structure or function. Consequently, PNA probes need to be evaluated for performance and optimization to thereby confirm whether they can be used to specifically and reliably detect a particular nucleic acid target sequence, particularly when the target sequence exists in a complex sample such as a cell, tissue or organism.
In summary, any method, kits or compositions which could improve the specificity, sensitivity and reliability of probe-based assays for the detection of microorganisms in samples of interest would be a useful advance in the state of the art particularly where the methods were uniformly applicable to probes of all or substantially all sequence variations. Moreover, the methods, kits or compositions would be particularly useful if they could provide for the rapid, reliable and sensitive multiplex analysis of samples for the presence of microoganisms such as bacteria.
This invention is directed to PNA probes, probe sets, methods and kits useful for detecting, identifying and/or quantitating one or more organisms of interest in a sample wherein the organisms are members of the bacterial species of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas fluorescens or organisms of a bacterial genus including the Salmonella genus, Bacillus genus or Pseudomonas genus. The PNA probes and probe sets of this invention comprise probing nucleobase sequences which allow for the specific detection of bacteria of a target species or genus.
The preferred probing nucleobase sequence of the probes of this invention are listed in Table 1, below. In preferred embodiments, PNA probes are organized into a set which is designed to detect, identify or quantitate certain species of bacteria, certain genus of bacteria or members of a defined set of species and genus of bacteria. In a most preferred embodiment, the probe set is suitable for the detection, identification and/or quantitation of USP bacteria (as defined herein).
This invention is further directed to a method suitable for detecting, identifying and/or quantitating one or more organisms of interest in a sample wherein the organisms are members of the bacterial species of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepatia, Pseudomonas fluorescens or organisms of a bacterial genus including the Salmonella genus, Bacillus genus or Pseudomonas genus. The method comprises contacting the sample with one or more PNA probes, wherein suitable probes are described herein. According to the method, the presence, absence or number of the one or more organisms of interest in the sample are then detected, identified or quantitated. Detection, identification and or quantitation is made possible by correlating the hybridization, under suitable hybridization conditions or suitable in-situ hybridization conditions, of the probing nucleobase sequence of a PNA probe to the target sequence with the presence, absence or number of the target organism of interest in the sample. This correlation is made possible by direct or indirect detection of the probe/target sequence hybrid.
In yet another embodiment, this invention is directed to kits suitable for performing an assay which detects the presence, absence or number of one or more organisms of interest in a sample. The kits of this invention comprise one or more PNA probes and other reagents or compositions which are selected to perform an assay or otherwise simplify the performance of an assay.
The PNA probes, probe sets, methods and kits of this invention have been demonstrated to be highly specific for the target organism(s) for which they are intended to detect. Moreover, the assays described herein are rapid (2-3 hours or less), sensitive, reliable and capable of both identification as well as enumeration of organisms listed in Table 1 in a single assay. Since probe-based analysis generically detects nucleic acid, the analysis of cells in culture is preferably used to distinguish between viable organisms and dead (non-viable) organisms, the presence of which are generally not considered to cause spoilage or contamination.
This invention is also directed to a multiplex PNA-fluorescent in-situ hybridization (FISH) assay. As a demonstration of the versatility of the PNA probes, probe sets, methods and kits of this invention, a PNA-FISH assay was multiplexed without any change to the protocol. The analysis was rapid, sensitive and reliable despite the substantial sequence variations of the probing nucleobase sequence of the PNA probes used for the different target organisms. Thus, Applicants have demonstrated (believed to be the first successful example) the feasibility of a multiplex PNA-FISH assay which can positively detect, identify and quantitate two or more target organisms in a single assay. Specifically, the multiplex assay as described in Example 10 provided a means for the detection, identification and quantitation of four target organisms using only three independently detectable moieties (fluorophores).
The PNA probes, probe sets, methods and kits of this invention are particularly useful for the detection of bacteria (pathogens) in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. The analysis of preferred beverages include soda, bottled water, fruit juice, beer, wine or liquor products. Suitable PNA probes, probe sets, methods and kits will be particularly useful for the analysis of raw materials, equipment, products or processes used to manufacture or store food, beverages, water, pharmaceutical products, personal care products dairy products or environmental samples.
Additionally, the PNA probes, probe sets, methods and kits of this invention are particularly useful for the detection of bacteria (pathogens) in clinical samples and clinical environments. Suitable PNA probes, probe sets, methods and kits will be particularly useful for the analysis of clinical specimens, equipment, fixtures or products used to treat humans or animals.